This invention relates to new macrocyclic chelants and metal chelates thereof, methods of preparing the chelants and metal chelates, and pharmaceutical compositions comprising the macrocyclic chelants and metal chelates. This invention relates particularly to the use of the new metal chelates as contrast agents in X-ray or CT, MRI imaging, and radiopharmaceuticals for the diagnosis of cardiovascular disorders, infectious disease and cancer. This invention also relates to new bifunctional chelants (BFCs) for attaching diagnostic metals and therapeutic isotopes to target-specific biomolecules such as proteins, peptides, peptidomimetics, and non-peptide receptor ligands. In addition, the macrocyclic chelants are useful for heavy metal detoxification.
Medical imaging modalities, such as MRI, X-ray, gamma scintigraphy, and CT scanning, have become extremely important tools in the diagnosis and treatment of various diseases and illness. Imaging of internal body parts relies on the contrast between the targeted organ and the surrounding tissues. The targeted organs or tissues are visible by the use of a particular metallopharmaceutical contast agent. In X-ray and CT diagnostics, increased contrast of internal organs, such as kidney, the urinary tract, the digestive tract, cardiovascular system, tumors, and so forth is obtained by administering a contrast agent which is substantially radiopaque. In conventional proton MRI diagnostics, increased contrast of internal organs and tissues may be obtained by administrating compositions containing paramagnetic metal species, which increase the relaxivity of surrounding water protons. In ultrasound diagnostics, improved contrast is obtained by administering compositions having acoustic inpedances different from that of blood and other tissues. In gamma scintigraphy, contrast of internal organ is obtained by the specific localization of a gamma ray emitting radiopharmaceutical.
Attachment of metal ions to biomolecules (BM) such as antibodies, antibody fragments, peptides, peptidomimetics, and non-peptide receptor ligands leads to useful target-specific diagnostic and therapeutic metallopharmaceuticals. These include fluorescent, radioactive and paramagnetic metal ions attached to proteins that can be used as probes in vivo in biological systems and in vitro in analytical systems as radioimmunoassays. For example, attachment of radionuclides to monoclonal antibodies that recognize tumor associated antigens provides radioimmunoconjugates useful for cancer diagnosis and therapy. The monoclonal antibodies are used as carriers of desired radioisotope to the tumor in vivo.
Radiopharmaceuticals can be classified into two primary classes: those whose biodistribution is determined exclusively by their chemical and physical properties; and those whose ultimate distribution is determined by receptor binding or other biological interactions. The latter class is often called target-specific radiopharmaceuticals. In general, a target specific radiopharmaceutical can be divided into four parts: a targeting molecule, a linker, a BFC, and a radionuclide. The targeting molecule serves as a vehicle, which carries the radionuclide to the receptor site at the diseased tissue or organ. The targeting molecules can be macromolecules such as antibodies; they can also be small biomolecules: peptides, peptidomimetics, and non-peptide receptor ligands. The choice of biomolecule depends upon the targeted disease or disease state. The radionuclide is the radiation source. The selection of radionuclide depends on the intended medical use (diagnostic or therapeutic) of the radiopharmaceutical. Between the targeting molecule and the radionuclide is the BFC, which binds strongly to the metal ion and is covalently attached to the targeting molecule either directly or through a linker. Selection of a BFC is largely determined by the nature and oxidation state of the metallic radionuclide. The linker can be a simple hydrocarbon chain or a long poly(ethylene glycol) (PEG), which is often used for modification of pharmacokinetics. Sometimes, an anionic poly (amino acid) is used to increase the blood clearance and to reduce the background activity, thereby improving the target-to-background ratio.
The use of metallic radionuclides offers many opportunities for designing new radiopharmaceuticals by modifying the coordination environment around the metal with a variety of chelants. The coordination chemistry of the metallic radionuclide will determine the geometry and solution stability of the metal chelate. Different metallic radionuclides have different coodination chemistries, and require BFCs with different donor atoms and ligand frameworks. For xe2x80x9cmetal essentialxe2x80x9d radiopharmaceuticals, the biodistribution is exclusively determined by the chemical and physical properties of the metal chelate. For target-specific radiopharmaceuticals, however, the xe2x80x9cmetal labelxe2x80x9d is not totally innocent because the target uptake and biodistribution will be affected by not only the targeting biomolecule but also the metal chelate and the linker. This is especially true for radiopharmaceuticals based on small molecules such as peptides due to the fact that in many cases the metal chelate contributes greatly to the overall size and molecular weight. Therefore, the design and selection of the BFC is very important for the development of a new radiopharmaceutical.
The same principle used for target-specific metallo-radiopharmaceuticals also applies to target-specific MRI contrast and ultrasound agents. Unlike the target-specific metalloradiopharmaceutical, where the excess unlabeled biomolecule can compete with the radiolabeled BFC-BM conjugate and block the docking of the radiolabeled receptor ligand, MRI and ultrasound contrast agents contain no excess unlabeled BFC-BM conjugate. Saturation of the receptor sites will maximize the contrast between the diseased tissues and normal tissue provided that the use of a relatively large amount of metal-BFC-BM chelate does not cause unwanted side effects.
For a therapeutic radiopharmaceutical or an MRI contrast agent, it is especially important to keep the metal chelate intact under physiological conditions, particularly in the presence of native chelators, such as transferrin, which have very high affinity for trivalent lanthanide metal ions. This requires the chelant to form metal chelate with high thermodynamic stability and kinetic inertness.
Several BFC systems such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepetaacetic acid (DTPA), as well as their derivatives, have been reported to form thermodynamically stable metal chelates. EDTA-based BFCs were first developed by Sunberg et al (Nature 1974, 250, 587) in the 1970s. Krejcarek and Tucker (Biochem. Biophys. Res. Commun. 1976, 77, 581) developed an activated DTPA analog via a mixed anhydride, which can be linked to proteins. Later, Hnatowich et al (Science 1983, 220, 613) used the cyclic anhydride of DTPA for the same purpose. These linear BFCs bond to a variety of metal ions like 111In or 90Y and form thermodynamically stable metal chelates. However, metal chelates of linear BFCs are kinetically labile, which contributes to the loss of radionuclide from the metal chelate and often leads to severe bone marrow toxicity. Gansow et al (Bioconjugate Chem. 1991, 2, 187; Inorg. Chem. 1986, 25, 2772) prepared a series of substituted DTPA analogs, which form metal chelates with improved solution stability.
Polyaza macrocycles have been widely used as chelants for a variety of transition metals. The macrocyclic polyaminocarboxylates such as 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetracetic acid (DOTA) and 1,4,8,11-tetraazacyclo-tetradecane-1,4,8,11-tetracetic acid (TETA) are known to form highly stable metal chelates due to their highly preorganized macrocyclic ligand framework. Their Gd chelates have been widely studied as MRI contrast agents. Examples include gadolinium complexes Gd-DOTA (Dotarem(trademark), Guerbet/France), Gd-HP-DO3A (ProHance(trademark), Bracco/Italy), and Gd-DO3A-butrol (Gadovist(trademark), Schering/Germany).
Macrocyclic chelants such as DOTA have also been used as BFCs for the radiolabeling of proteins (antibodies or antibody fragments) and peptides with various diagnostic and therapeutic radionuclides (such as 111In and 90Y). Meares and coworkers were the first to synthesize macrocyclic BFCs (Anal. Biochem. 1985, 148, 249; Nucl. Med. Biol. 1986, 13, 363; Inorg. Chem. 1987, 26, 3458), which form 67Cu and 90Y chelates with high thermodynamic stability and kinetic inertness. Macrocyclic chelants with three-dimensional cavities are of particular interest because of the high stability of the metal chelates, the substantial selectivity for certain metal ions, either by enforcing a specific spatial arrangement of donor atoms or by introducing different donor atoms into the ligand backbone, and their capability to adopt a preorganized conformation in the unchelated form. The higher the degree of preorganization of an unchelated ligand, the more stable the complex is.
Preorganization of a polydentate chelant results in not only the high thermodynamic stability but also the increased kinetic inertness of its metal chelate. This has been exemplified by the fact that the half-life for [Gd(DOTA]xe2x88x92in 0.1 M HCl is 60.2 h and 2000 years at pH=6.4 while the complex [Gd(DTPA]2xe2x88x92having comparable thermodynamic stability decomposes rapidly under acidic conditions with a half-life of xcx9c1.0 min. The highly preorganized macrocyclic framework of DOTA forces four acetate chelating arms to adopt such a conformation that the metal ion can be completely wrapped by an N4O4 donor set. At the same time, this also makes it more difficult for the coordinated acetate to be dissociated from the metal center. Therefore, preorganization should be an important factor in the design of new BFCs for the radiolabeling of biomolecules.
Generally, there are three possible approaches to attach a biomolecule to a DOTA-based chelant. In the first approach, the attachment is at one of the carbon atoms of the macrocyclic chelator backbone. In principle, this will result in formation of eight possible isomers when coordinated to the lanthanide metal ion. In the second approach, the linker is attached to the methylene-carbon atom of one of four acetate chelating arms, which may also result in formation of eight possible isomeric forms. In both approaches, the conjugation of the biomolecule does not lead to a significant change in the thermodynamic stability and kinetic inertness of the metal chelate as compared to those of the DOTA chelate. In the third approach, the biomolecule is conjugated to one of the four acetate groups via a COxe2x80x94N amide bond. Compared to the carboxylate-O, the carbonyl-O is a relatively weak donor for yttrium and lanthanide metal ions. This often leads to the lower thermodynamic stability of the corresponding metal chelate. However, the kinetic inertness of its metal complex remains relatively unchanged.
In U.S. Pat. No. 4,678,667, Meares et al disclosed a copper chelate conjugate for diagnostic or therapeutic applications. The bifunctional macrocyclic chelants include substituted DOTA, TETA, TRITA, HETA. The linker is at least 8-atom in length and the attachment position of the linker is on the carbon atom of the polyamine macrocycle. In U.S. Pat. No. 5,428,156 disclosed a method of producing DOTA, TETA, DOTA-NHS(NHS=N-hydroxysuccinimide) and TETA-NHS esters for conjugation of biomolecule. Meares et al (WO 95/26206 and U.S. Pat. No. 5,958,374) also disclosed a method for preparing a radionuclide-labeled chelating agent complex. It specifically disclosed DOTA(Gly)3-L-(p-isothiocyanato)-Phe-amide as the BFC. The pendant linkers also include xe2x80x94CH2COxe2x80x94(AA)mxe2x88x92(AA-Phe-Gly), where AA represents an amino acid diradical, more preferably the glycine diradical xe2x80x94NHCH2COxe2x80x94. Gansow et al (WO 89/11475, WO 91/14458, U.S. Pat. Nos. 4,923,985 and 5,428,154) disclosed a process of making 4-aminophenyl-DOTA and its use a BFC for the radiolabeling of biomolecules such as antibody. Parker et al (WO 87/05030, WO89/01476, EP 0382583B1 and EP 0382583A1) disclosed a series of DOTA analogs as BFCs, which are coupled with biomolecules such as a protein, especially antibodies, peptides or carbohydrates to form conjugate compounds. The linker and conjugation group is attached to either one of the four acetate chelating arms or one of the carbon atom of the macrocyclic backbone. Watson, et al (WO 90/12050 and WO93/06868) disclosed polychelants and their metal chelates useful in diagnostic imaging and in radiotherapy. The macrocyclic chelant moieties are linked to the backbone moiety (dendrimer or polylysine) via an amide-bond. In U.S. Pat. No. 5,053,053, Dean et al also disclosed a series of DOTA and DO3A analogs as BFCs. For DO3A-based BFCs, the conjugation group is connected to a linker attached to one of the four amine-nitrogen atoms. For DOTA derivatives, the linker group is connected to either one of carbon-atoms on the macrocyclic backbone or the methylene-carbon atom of one of the four acetate chelating arms. Tweedle, et al (EP 0292689 A2/A3; U.S. Pat. Nos. 4,885,363, 5,474,756, and 5,846,519) disclosed metal chelates, particularly those of neutral charge, for MRI contrast imaging. It also disclosed DO3A analogs as BFCs for the radiolabeling of biomolecules. Kruper et al (U.S. Pat. Nos. 5,310,535 and 5,739,323) disclosed the DOTA analogs as BFCs for the radiolabeling of proteins. The linker is connected to the acetate chelating arm and the conjugation group is on a benzene ring. It was shown that the DOTA monoamide has better kinetic inertness because of less bone uptake. Kubomura et al (AU9335519 and EP 0565930A1) disclosed the use of DO3A-CH2CONHCH2CH2NH2 as the BFC, and the metal chelates of BFC-BM conjugates as diagnostic or therapeutic pharmaceuticals. Gozzini et al (WO 97/32862) disclosed a new class of polychelants, their chelates with metal ions and their physiologically acceptable salts, which can be used, either as they are or in association or formulation with other components, for diagnostic imaging in general or specific contrast agents for specific tissues, organs or body compartments. It specifically discloses DOTA as the BFC, and a process of making these macrocyclic chelants with DO3A-CH2CONHCH2CH2CHO and poly(amino acids) as key intermediates. Wilson et al (U.S. Pat. No. 5,756,065) also disclosed DOTA analogs as BFCs. The conjugation group is attached to a benzene ring and the linker group is connected to one of the four acetate chelating arms. Almen et al. (U.S. Pat. No. 5,348,954) discloses heterocyclic chelating agents for use in heavy metal detoxification. Watson (U.S. Pat. No. 5,914,095) also discloses polychelants for use in metal detoxification.
The present invention provides macrocyclic chelants containing a substituted pyridinone moiety. These macrocyclic chelants are unique for several reasons. The hydroxy group of the pyridinone heterocycle has a higher pKa value than the carboxylic group and the hydroxy-O is a better donor atom than the corresponding carbonyl-O atom in a DOTA-biomolecule conjugate when bonded to xe2x80x9chardxe2x80x9d trivalent lanthanide metal ions. These macrocyclic chelants will form anionic metal chelates with higher hydrophilicity, which is beneficial for improved pharmacokinetics. The pyridinone binding unit is bidentate, and is available to form a xe2x80x9cpre-chelatexe2x80x9d before the metal ion goes into the coordination cavity of the macrocycle. This, in return, will result in improved radiolabeling kinetics. Like DOTA, the macrocyclic chelants are expected to form stable complexes with trivalent metal ions such as In3+, Y3+, Sm3+, Gd3+, Dy3+, Ho3+, Yb3+, and Lu3+. Unlike phenols, the pyridinone ring is radiolytically stable, which is very important to maintain the solution stability of therapeutic radiopharmaceuticals.
The present invention also provides macrocyclic chelants containing a succinimide or phthalimide functional group. The succinimide or phthalimide group is connected to one of the four amine-nitrogen atoms of the macrocycle via a C1-C3 alkylene linker in such a way that the carbonyl-O atom of the succinamide or phthalimide group is available to coordinate the lanthanide metal ions to form 8- or 9-coordinated metal chelates. Unlike macrocyclic chelants with substituent(s) on the acetate chelating arm or macrocyclic backbone, macrocyclic chelants containing a succinimide or phthalimide group form metal chelates with only two isomers. Due to the presence of DO3A chelating unit, macrocyclic chelants containing a succinimide or phthalimide group will form lanthanide metal chelates with high thermodynamic stability and kinetic inertness.
The present invention also provides macrocyclic chelants containing a linker group, such as phosphotriester, phosphodiester, phosphodiestermonoamide-, and phosphomonoester-diamide. Like carbonyl-O and carboxylate-O atoms, the phosphonyl-O and phosphonate-O are also good donor atoms for xe2x80x9chardxe2x80x9d, trivalent lanthanide metal ions. These macrocyclic chelants form either neutral or anionic metal chelates with trivalent metal ions such as In3+, Y3+, Sm3+, Gd3+, Dy3+, Ho3+, Yb3+, and Lu3+.